Use of heat shock protein 70 preparations in vaccination against cancer and infectious disease

ABSTRACT

The use of cognate heat shock protein 70-peptide complex to elicit an immune response against cancer and viral, bacterial and other infectious agents.

This is a continuation of application No. 08/180,685, filed Jan. 13,1994 now U.S. Pat. No. 5,997,873 which is incorporated by referenceherein in its entirety.

This invention was made with Government support under National Instituteof Health Grant No. CA44786 (GCO Project #88-416 PHA; Fund #G5-204X).The Government has certain rights in this invention.

FIELD OF THE INVENTION

This invention relates to a method of using heat shock protein 70preparations obtained from tumor cells or cells infected with a virus orother agent in order to elicit an immune response against the tumor,virus or other agent.

BACKGROUND OF THE INVENTION

The observation that inbred mice and rats can be immunized against theirown tumors or tumors of the same genetic background have led to ahypothesis that tumor-specific antigens exist. In essence, these studiesshowed that mice vaccinated with inactivated cancer cells are immune tosubsequent challenges of live cancer cells. The phenomenon was shown tobe individually tumor-specific, in that mice were immune specifically tothe tumors used to immunize them and not to other tumors. Thedemonstration of immunogenicity of cancer cells led to a search for thecancer-derived molecules which elicit resistance to tumor challenges.The general approach in these experiments was to fractionatecancer-derived proteins and test them individually for their ability toimmunize mice against the cancers from which the fractions wereprepared.

One of the major difficulties in cancer immunotherapy has been thepossibility that similar to the situation among animal cancers, eachhuman cancer is different from all other cancers, i.e., human cancers,like cancers of experimental animals, are antigenically distinct.Clearly, there is some recent evidence for existence of common humantumor antigens (Kawakami et al., 1991, Darrow et al., 1989), and thisaugurs well for prospects of cancer immunotherapy. Nonetheless, in lightof the overwhelming evidence from experimental and human systems, it isreasonable to assume that at the very least, human tumors would showtremendous antigenic diversity and heterogeneity.

The prospect of identification of the immunogenic antigens of individualtumors from cancer patients (or even of ‘only’ several different typesof immunogenic antigens in case the antigens are shared), is daunting tothe extent of being impractical. Numerous studies on vaccination againstinfectious diseases have shown that it is necessary to first identifyand characterize the immunogenic antigens.

For the reasons described above, such a strategy is impractical forvaccination or other forms of immunotherapy against human cancers. Thus,there is a need to develop alternate methods for obtaining antigenicpreparations which do not require such daunting identification ofspecific antigens from tumors of individual patients and avoids thedifficulties and hazards associated with attenuation and inactivation ofviruses.

SUMMARY OF THE INVENTION

This invention relates to an immunogenic composition comprisingcomplexes of heat shock protein 70 and antigenic peptides derived fromtumor cells or cells infected with a virus, bacteria or other agent.This invention also relates to a method of eliciting an immune responsein a mammal comprising the steps of isolating heat shock protein70-peptide complex from tumor cells or cells infected with a virus,bacteria or other agent and administering the heat shock protein70-peptide complex to the mammal in an amount effective to elicit animmune response. The claimed invention provides a novel method ofeliciting antigen-specific cellular immunity against tumors, endogenousantigens, bacterial and viral antigens.

This invention further relates to a method of preparing a heat shockprotein 70-peptide complex capable of eliciting an immune response in amammal comprising the steps of obtaining tumor cells from the mammal orcells which are infected with a virus, bacteria or other infectiousagent, preparing an aqueous cell extract, purifying the extract throughcolumn chromatography and harvesting a heat shock protein 70-peptidecomplex in the absence of adenosine triphosphate (ATP).

This invention additionally relates to a method of preparing anantigenic peptide composition comprising obtaining cells from a mammalwherein the cells are tumor cells or cells infected with a virus,bacteria or other infectious agent, harvesting a heat shock protein70-peptide complex from the cells wherein the heat shock protein70-peptide complex is prepared in the absence of ATP and separatingpeptides from the heat shock protein 70-peptide complex, wherein theseparated peptides are capable of eliciting an immune response in themammal.

This invention also relates to an immunogenic composition comprisingcomplexes of heat shock protein 70 and antigenic peptides derived fromtumor cell lines or cell lines infected with a virus or bacteria. Theinvention further relates to a method of preparing a heat shock protein70-peptide complex capable of eliciting an immune response in a mammalcomprising preparing an aqueous cell extract from tumor cell lines orcell lines infected with a bacteria or virus, purifying the extractthrough column chromatography and harvesting a heat shock protein70-peptide complex in the absence of ATP.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-B depict SDS-PAGE followed by silver staining of (a) purifiedhsp 70 preparations from Meth A ascitic cells and (b) immunoblot of hsp70 preparations in (a) with anti-heat shock protein antibody.

FIGS. 2A-C depict kinetics of tumor growth in mice immunized with 9 μg,6 μg, and 3 μg of hsp 70-peptide complex.

FIGS. 3A-G depict kinetics of tumor growth in mice immunized with hsp 70preparations purified by conventional chromatography and by ATP-agarosechromatography.

FIGS. 4A-B depict kinetics of growth of the CMS4 tumor in mice immunizedwith (a) Meth A ascitic cell lysate or (b) hsp 70-peptide complexderived from CMS4 cell line.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

It has been found that vaccination of mice with heat shock protein 70(hsp 70) preparations derived from methylcholanthrene-induced sarcoma(Meth A), e.g., a hydrocarbon-induced sarcoma, but not from normaltissues renders the mice immune to a substantial challenge with Meth Asarcoma. This immunity is tumor-specific. It has also been found thathsp 70 loses its antigenicity upon treatment with ATP. Such treatment isknown to result in dissociation of hsp 70 from a spectrum of peptides.Considering that there are no known differences in the structure of hsp70 per se between normal and cancer cells, and that hsp 70 binds a widespectrum of peptides in an ATP-dependent manner, it appears that theantigenicity of hsp 70 derives, not from hsp 70 per se, but fromassociated peptides. Thus, the claimed invention provides a novel methodof utilizing the peptide binding property of hsp 70 comprising isolatinga heat shock protein 70-peptide complex from tumor cells or cellsinfected with a virus, bacteria or infectious agent and administeringthe heat shock protein 70-peptide complex to a mammal in an amounteffective to elicit an immune response. In this regard, immunizationwith this hsp 70-peptide complex is believed to elicit a CD4+ and CD8+ Tcell response and is therefore useful in promoting cell-mediatedimmunity which is particularly important as a defense against viral andbacterial infections or tumors.

In addition, the present invention provides for a method for purifyinghsp 70 which leaves intact the association between hsp 70 and theantigenic peptides which it “chaperones”. This method, set forth indetail in the example sections which follow, may be applied to eithertumor cells and virus or bacteria-infected cells to the same effect. Inthe case of tumor cells, hsp 70 in association with tumor-specificantigenic peptides is purified, and in the case of virus-infected cells,hsp 70 in association with viral antigenic peptides is purified, and inthe case of bacteria-infected cells, hsp 70 in association withbacterial antigenic peptides is purified. In this regard, purificationof the hsp 70 associated with antigenic peptides must be performed inthe absence of adenosine triphosphate (ATP), which appears to displacethe antigenic peptides associated with hsp 70.

The present invention further provides for the purification andisolation of antigenic peptides which are associated (or “chaperoned”)by hsp 70 in virus or bacteria-infected or tumor cells. A non-denaturingmethod may be used to elute chaperoned peptides from the hsp 70-peptidecomplex described above. In a specific, non-limiting embodiment of theinvention, an hsp 70-peptide complex (e.g., as prepared in the examplesections which follow, or from the same methods as applied to virus orbacteria-infected cells) may be centrifuged through a Centricon 10assembly in order to remove any low molecular weight material looselyassociated with it. The large molecular weight fraction may be recoveredand analyzed by SDS-PAGE while the low molecular weight material may beanalyzed by HPLC, as described infra. The hsp 70 preparation in thelarge molecular weight fraction may be incubated with ATP at a finalconcentration of about 10 mM at room temperature for 30 minutes andcentrifuged through Centricon 10 as before. The two fractions may berecovered, and the ATP treatment of the large molecular weight hsp 70fraction may be repeated two or more times. The lower molecular weightfractions may then be pooled, concentrated by evaporation in a Speed Vacand then dissolved in 0.1% trifluoroacetic acid (TFA). This material maythen be applied to a VYDAC C18 reverse phase HPLC columnpre-equilibrated with 0.1% TFA. The bound material may then be eluted ata flow rate of about 0.8 ml/min by a linear gradient of 0 to 79.9%acetonitrile in 0.1% TFA. The ultraviolet light absorbance at 210 nm maybe monitored to identify fractions containing antigenic peptide.Antigenic peptides prepared in this manner may be used in immunogeniccompositions which may be used to elicit immunity in a mammal in need ofsuch treatment. It may, in certain circumstances, be desirable toadminister such peptides linked to or otherwise associated with acarrier molecule, so as to promote immunity.

The present invention also provides for immunogenic compositions whichcomprise either hsp 70-peptide complex or antigenic peptides. Suchcompositions may further comprise a suitable carrier such asphosphate-buffered saline (5 mM Na phosphate buffer, 150 mM NaCl, pH7.1) or other physiologically compatible solution. The immunogeniccomposition may optionally comprise one or more adjuvants. Suitableadjuvants include, but are not limited to, pluronic tri-blockcopolymers, muramyl dipeptide and its derivatives, detoxified endotoxin,saponin and its derivatives such as QS-21 and liposomes. The presentinvention further envisages sustained release formulations in whichantigen is released over a protracted period of time. In preferred,non-limiting embodiments of the invention, the amount of hsp 70-peptidecomplex administered may be about 50-1000 micrograms/kg body weight ofthe mammal, most preferably 100-250 micrograms/kg body weight, perimmunization, and in particular, about 7.5 mg to 18.75 mg for anapproximately 75 kilogram human subject. The quantities of hsp70-peptide complex administered to human subjects may not beextrapolated directly from the amounts used in mice, as would be thecase for antibiotics and metabolic drugs. Because of the immune system'sability to amplify responses extremely efficiently, smaller quantitiesof hsp 70-peptide complex may be required to immunize human subjectsthan would be expected from a direct extrapolation from mice. Further,the quantities may vary depending upon the adjuvant formulation whichmay be administered along with the hsp 70-peptide complex.

The immunogenic compositions of the invention may be administered inimmunogenic amounts to subjects in need of such treatment using standardprotocols, which would include, but not be limited to, intramuscular,subcutaneous, intradermal, intraperitoneal, intravenous, intravaginal,intrarectal, oral, sublingual, transcutaneous, and intranasaladministration. It may be desirable to provide a series of immunizationsin order to optimize the immune response.

The hsp 70-peptide complex can be prepared from tumor cells, including,but not limited to, adenocarcinomas, colon carcinoma, melanoma, breastcarcinoma, leukemia, lymphoma, sarcomas (including fibrosarcoma andosteosarcoma), gastric carcinoma, glioblastoma, astrocytoma, bladdercarcinoma, pleural mesothelioma, oat cell carcinoma, and bronchogeniccarcinoma, as well as tumors induced by chemical carcinogens orradiation. Chemical carcinogens include carcinogens associated withcigarette smoking, such as hydrocarbons and carcinogenic air, food,cosmetic or other pollutants.

The hsp 70-peptide complex can also be prepared from virus-infectedcells where the virus may be influenza, varicella, herpes simplex I orII, HIV-I or HIV-II, hepatitis A, B or C, adenovirus, measles, mumps,etc.

The hsp 70-peptide complex may also be prepared from bacteria-infectedcells including, but not limited to, cells infected with bacteriacausing tuberculosis, gonorrhea, typhoid, meningitis, osteomyelitis,meningococcal septicemia, endometritis, conjunctivitis, peritonitis,pyelonephritis, pharyngitis, septic arthritis, cellulitis, epiglottitis,salpingitis, otitis media, shigella dysentery, gastroenteritis, etc.

The hsp 70-peptide complex can also be prepared from tumor cell lines orcell lines infected with a virus or bacteria. In addition, the hsp70-peptide complex may be prepared from viral gene transfected cells.

Immunization with an hsp 70-peptide complex offers a number ofsignificant and unique advantages over other methods of immunizationagainst viruses, bacteria or cancer. Hsp 70 protein-peptide complexcarries a variety of immunogenic peptides derived from the cells fromwhich it is isolated. Thus, immunization with an hsp 70-peptide complexobviates the necessity for isolation and characterization of antigenicmolecules. In addition, immunization with biochemically undefined tumoror other extracts inevitably carries the risk of inoculating the mammalrecipient with potentially transforming or immunosuppressive agents suchas transforming DNA or tumor growth factor beta (TGFB). Immunizationwith purified hsp 70-peptide complex eliminates these risks. Moreover,it has been found that immunization with hsp 70-peptide complex elicitssignificant tumor immunity without the use of adjuvants. While adjuvantswhich may further potentiate the immunity elicited by the hsp 70-peptidecomplex may be sought, their availability is not a pre-condition for asignificant protective response. This is a very significant advantagefor human subjects because the availability of adjuvants for human useis rather limited.

The claimed invention is one of the very few methods of vaccinationwhich elicit cellular immunity without the use of live (attenuated orotherwise) agents. The immunogenic compositions prepared from hsp70-peptide complex in accordance with the invention are an idealvaccination means for infections for which either the protectiveimmunogenic epitopes are yet undefined, where binding to a singleepitope may not be sufficient for eliciting immunity, or where theinfectious agent is so highly variable (in a population, season orindividual-specific manner) that the prospect of identifying theimmunogenic epitopes for each variant is simply impractical. Inaddition, the hsp 70-peptide complex has a number of different peptidesassociated with it, which potentially may include a number of differentantigens capable of binding to a variety of epitopes. As hsp 70molecules are non-polymorphic, i.e., show no allelic diversity, eventhough there are several families, they are capable of binding theentire spectrum of antigenic peptides regardless of the MHC haplotype ofa given cell. Thus, an hsp 70-peptide complex isolated from cells of anygiven haplotype may be used to vaccinate individuals of otherhaplotypes. Moreover, the recent recurrence of antibiotic-resistantstrains of a number of infectious diseases presently treated byantibiotics and metabolic drugs such as tuberculosis, highlights theneed for a general method as provided by the claimed invention which canbe rapidly mobilized against a variant without having to define itsmolecular characteristics.

The novel ability of hsp 70-peptide complex in accordance with theinvention to elicit an immune response is illustrated in the followingexamples.

EXAMPLE 1 Preparation of Purified hsp 70-peptide Complex

BALB/cJ mice (viral antigen free) were obtained from Jacksonlaboratories and were maintained in virus-free mouse facilities. Tumorcells were injected intraperitoneally in 0.2 ml volume as described inSrivastava et al., Proc. Natl. Acad. Sci., vol. 83, pp. 3407-3411 (May,1986). Meth A ascites cells were collected after 7 days and weresuspended at a density of 10⁶ cell/ml in Dulbecco's Modified EaglesMedium (DMEM) without methionine, containing 10% dialyzed fetal calfserum. Cells were cultured in the absence of methionine for 4 hours inorder to deplete methionine pools. They were then re-suspended in freshmethionine-free medium containing Trans-Label (which contains³⁵S-methionine 100 μCi/ml) for one hour and harvested and a 40 ml Meth Acell pellet was derived. The 40 ml Meth A cell pellet was homogenized in120 ml hypotonic buffer (30 mM NaHCO₃, pH 7.1, 0.5 mM phenyl methylsulfonyl fluoride (PMSF)) and a 100,000 g supernatant obtained. This wasapplied to a Concanavalin A-Sepharose column in presence of 2 mM Ca⁺⁺and the unbound material was dialyzed against 10 mM tris-acetate pH 7.5,10 mM NaCl, 0.1 nM EDTA. This fraction was resolved on a Mono QPharmacia FPLC system equilibrated with 20 mM tris-acetate pH 7.5, 20 mMNaCl, 0.1 mM EDTA, 15 mM 2-mercaptoethanol. The proteins were eluted bya 20 mM to 500 mM NaCl gradient. Fractions (1 ml) were collected andtested by SDS-PAGE. Hsp 70 containing fractions were identified bymolecular weight and by immunoblotting with anti-hsp 70 monoclonalantibody.

The hsp 70 containing fractions were pooled and precipitated withincreasing saturation levels of ammonium sulfate. Hsp 70 wasprecipitated at 50%-70% ammonium sulfate saturation. The later fractionsin this process were shown to be homogeneous by silver staining and wereused for immunization of mice. The left lane in FIG. 1A shows theSDS-PAGE profile of a purified hsp 70 fraction. The purified hsp 70fraction obtained in the absence of ATP, shown in the left lane of FIG.1A, was immunoblotted on nitrocellulose and probed with a group hsp 70monoclonal antibody N27F3-4 (Stress Gen product # SPA-820) (FIG. 1B).The right lane in FIG. 1A is the SDS-PAGE profile for precipitatedfractions collected as described above but which were additionallypassed through an ATP-agarose column and eluted in the presence of 3 mMATP. The right lane in FIG. 1(a) is the SDS-PAGE profile forprecipitated fractions collected as described above but which wereadditionally passed through an ATP-agarose column and eluted in thepresence of 3 mM ATP.

For purification of hsp 70 from liver, the 100,000 g supernatant wasfirst applied to a Blue Sepharose column in order to remove albumin.

EXAMPLE 2 Tumor Immunogenicity of hsp 70-peptide Complex

The biochemically homogeneous hsp 70 preparations obtained in theabsence of ATP from the BALB/cJ fibrosarcoma Meth A or from normaltissue, as described above, were used to immunize BALB/cJ mice twice atweekly intervals. Immunization was carried out in 200 μl volumesubcutaneously. Mice were challenged with 70,000 live Meth A cellsintradermally one week after the second immunization. The kinetics oftumor growth is shown in FIGS. 2A-C. Each line represents the kineticsof tumor growth in a single mouse. Tumors grew progressively in allunimmunized mice, but there was significant protection from tumor growthin hsp 70-peptide complex immunized mice. This effect wasdose-dependent. Two injections of 3 μg of hsp 70-peptide complex eachdid not immunize mice against Meth A, while two injections of 9 μg eachconferred complete protection to all vaccinated mice. As hsp 70 is aubiquitous protein, present in normal tissues as well as in tumors, hsp70 preparations from normal liver and spleens were tested forimmunogenicity in the same manner. No protective effect of hsp 70derived from normal tissues was observed at any of the three dosestested (see FIGS. 2A-C).

EXAMPLE 3 Tumor Specificity of Meth A Derived hsp 70-peptide Complex

In order to determine the tumor specificity of the Meth A derived hsp70-peptide complex, mice were challenged with antigenically distinctmethylcholanthrene-induced BALB/cJ sarcomas CMS4 and CMS5 in accordancewith the procedure described in Example 2. The results are shown inTable 1.

TABLE 1 Specificity of immunity elicited by immunity with hsp 70 derivedfrom Meth A sarcoma Number of tumor cells used for Tumor used forchallenge Mice challenge Meth A CMS5 CMS4 Immunologically 5 × 10⁴  5/5¹4/5 N.D. naive mice 1 × 10⁵ 5/5 5/5 5/5 Mice immunized 5 × 10⁴ 0/5 5/5N.D. with Meth A hsp 70² 5 × 10⁵ 0/5 5/5 5/5 ¹Number of mice in whichthe tumors grew per total number of mice challenged. ²Mice wereimmunized with 10 μg of Meth A hsp 70, twice at weekly intevals andchallenged with 50,000 Meth A cells. The mice rejected the Meth Asarcoma and were challenged with other sarcomas.

As can be seen from Table 1, mice immunized with Meth A derived hsp70-peptide complex remained sensitive to challenge with antigenicallydistinct methylcholanthrene-induced BALB/cJ sarcomas CMS4 and CMS5.

EXAMPLE 4 Immunogenicity of hsp 70 Purified in the Presence of ATP

The hsp 70 purified in the presence of 3 mM ATP as described in Example1 was tested for immunogenicity in accordance with the proceduredescribed in Example 2. The results are shown in FIGS. 3A-G. The Figureshowed tumor growth for mice not immunized (FIG. 3A), tumor growth formice immunized with Meth A derived hsp 70-peptide complex purified inthe absence of ATP (FIGS. 3B-D), and in the presence of ATP (i.e.,peptide depleted) (FIGS. 3E-G). Mice vaccinated with Meth A derived hsp70-peptide complex purified in the absence of ATP were significantlyprotected against tumor challenge, but the mice vaccinated with theATP-eluted Meth A derived hsp 70-peptide complex were not.

These data suggest that the- immunogenicity of the Meth A derived hsp70-peptide complex derives from the antigenic peptides associated withit. The peptides are most likely derived from cellular proteins byproteolytic degradation during antigen presentation by majorhistocompatibility complex (MHC) Class I and Class II proteins. The hsp70 molecules encounter peptides in the endoplasmic reticulum, endosomesand in the cytosol. While not wishing to be confined to a specifictheory, the peptides generated in tumor cells must clearly differ fromthose generated in normal tissues because of the tumor associatedmutations, and may therefore result in the difference in theantigenicity of tumor versus normal cell derived hsp 70-peptide complex.

EXAMPLE 5 Tumor Immunogenicity of hsp 70-peptide Complex Derived fromCell Lines

In order to determine the tumor immunogenicity of an hsp 70-peptidecomplex, cells from the CMS4 cell line were cultured in 10% fetal calfserum in Roswell Park Memorial Institute (RPMI) medium. A 4 ml CMS4 cellpellet was derived. The 4 ml CMS4 cell pellet was homogenized in 12 mlhypotonic buffer (30 mM NaHCO₃, pH 7.1, 0.5 mM PMSF) and a 100,000 gsupernatant obtained. This was applied to a Concanavalin A-Sepharosecolumn in presence of 2 mM Ca⁺⁺ and the unbound material was dialyzedagainst 10 mM tris-acetate pH 7.5, 10 mM NaCl, 0.1 mM EDTA. Thisfraction was resolved on a Mono Q Pharmacia FPLC system equilibratedwith 20 mM tris-acetate pH 7.5, 20 mM NaCl, 0.1 mM EDTA, 15 EM2-mercaptoethanol. The proteins were eluted by a 20 mM to 500 mM NaClgradient. Fractions (1 ml) were collected and tested by SDS-PAGE. Hsp 70containing fractions were identified by molecular weight and byimmunoblotting with anti-hsp 70 monoclonal antibody. The hsp 70containing fractions were pooled and precipitated with increasingsaturation level of ammonium sulfate. Hsp 70 was precipitated at 50%-70%ammonium sulfate saturation. The later fractions in this process-wereshown to be homogeneous by silver staining and were used forimmunization of mice.

The purified hsp 70 preparation obtained from the CMS4 cell line asdescribed above or lysate from Meth A cells were used to immunizeBALB/cJ mice. The mice were immunized with 9 μg of either the purifiedhsp 70 preparation obtained from the CMS4 cell line or Meth A lysatetwice at weekly intervals. Immunization was carried out in 200 μl volumesubcutaneously. Mice were challenged with 50,000 live CMS4 cellsintradermally one week after the second immunization. The kinetics oftumor growth is shown in FIGS. 4A-B. Each line represents the kineticsof tumor growth in a single mouse. Tumors grew progressively in miceimmunized with the Meth A lysate (FIG. 4A), but there was significantprotection from tumor growth in mice immunized with hsp 70-peptidecomplex derived from the CMS4 cell line (FIG. 4B).

Although the invention has been described herein with reference tospecific embodiments, many modifications and variations therein willreadily occur to those skilled in the art. Accordingly, all suchvariations are included within the intended scope of the invention.

I claim:
 1. A composition comprising an amount of a purified populationof peptides, wherein said purified population of peptides is produced bya method comprising the steps of: a) isolating a population ofnon-covalently associated heat shock protein 70-peptide complexes frommammalian tumor cells in the absence of ATP; b) eluting the peptidesfrom said population of complexes to produce dissociated peptides; andc) recovering the dissociated peptides.
 2. The composition of claim 1,wherein in step (a) the population of complexes is isolated from humantumor cells.
 3. A composition comprising an amount of a purifiedpopulation of peptides, wherein said population of peptides ischaracterized as being present as non-covalent complexes with aplurality of heat shock protein 70 polypeptides in mammalian tumorcells.
 4. The composition of claim 3, wherein the mammalian tumor cellsare from a cell line.
 5. The composition of claim 2 or 3, wherein themammalian tumor cells are tumor tissue from a patient.
 6. Thecomposition of claim 2 or 3, wherein the mammalian tumor cells arecancer cells of a cancer selected from the group consisting ofadenocarcinoma, colon carcinoma, melanoma, breast carcinoma, leukemia,lymphoma, sarcoma, gastric carcinoma, glioblastoma, astrocytoma, bladdercarcinoma, pleural mesothelioma, oat cell carcinoma, and bronchogeniccarcinoma.
 7. The composition of claim 2 or 3, wherein the mammaliantumor cells are derived from mammalian tumor tissue induced by achemical carcinogen.
 8. The composition of claim 7, wherein themammalian tumor tissue is hydrocarbon-induced.
 9. The composition ofclaim 8, wherein the mammalian tumor tissue ismethylcholanthrene-induced.
 10. The composition of claim 1, 2 or 3,wherein said heat shock protein 70 is a human heat shock protein
 70. 11.The composition of claim 1, 2, 3, or 4 further comprising an adjuvant.12. The composition of claim 11, wherein the adjuvant is selected fromthe group consisting of a pluronic tri-block copolymer, muramyldipeptide, detoxified endotoxin, saponin, QS-21, and a liposome.